This project is aimed at elucidating the mechanisms by which the Nef and Vpu proteins of human immunodeficiency virus 1 (HIV-1) downregulate CD4 in the viral host cells, T-lymphocytes and macrophages. HIV-1 Nef is a 27-kDa myristoylated accessory protein that is produced at high levels early during infection. Nef is an important determinant of pathogenicity, as demonstrated by the finding that some long-term non-progressors (i.e., untreated infected persons who do not develop symptoms of AIDS for 10 years or longer) carry an HIV-1 strain with inactivating mutations of the Nef gene. An understanding of Nef function could thus provide new avenues for therapeutic intervention. Nef has many effects in the infected cells, the best characterized of which is the downregulation of CD4. Together with chemokine receptors, CD4 serves as a co-receptor for HIV-1 entry into cells. The downregulation of CD4 by Nef is thought to prevent superinfection and increase the release of infectious particles, thus explaining the higher virulence of Nef-carrying HIV-1 strains. Previous work from our laboratory demonstrated that the downregulation of CD4 by HIV-1 Nef requires clathrin and the heterotetrameric, clathrin-associated adaptor protein-2 (AP-2) complex in the host cells. In addition, we demonstrated that this requirement stemmed from a direct interaction of Nef with AP-2 involving a dileucine and a diacidic motif in Nef and the alpha and sigma2 subunits of AP-2. Through mutational analyses, we mapped the binding site for both Nef motifs on alpha and sigma2. Since clathrin and AP-2 mediate endocytosis from the plasma membrane, these findings indicated that Nef downregulates CD4 by accelerating its internalization form the surface of host cells. This past year, we discovered a new function of Nef: the targeting of internalized CD4 to the multivesicular body (MVB) pathway for eventual degradation in lysosomes. Following Nef-induced internalization, CD4 is degraded in lysosomes, leading to a profound reduction of total CD4 levels in the cell. The lysosomal degradation of some signaling receptors depends on ubiquitination-dependent targeting to the multivesicular body (MVB) pathway. We found that both Nef and CD4 are ubiquitinated on lysine residues. However, Nef expression does not change the extent and pattern of CD4 lysine ubiquitination. Moreover, downregulation is independent of lysine ubiquitination of both Nef and CD4. Nonetheless, Nef-induced CD4 downregulation is dependent on the ESCRT complexes that mediate targeting to intraluminal vesicles of MVBs. These results indicate that Nef targets internalized CD4 for lysine-ubiquitination-independent delivery to the MVB pathway en route to lysosomes. At later stages of infection, the HIV-1 genome directs expression of a second protein, Vpu, that downregulates CD4. Vpu is a type I integral membrane proteins that inserts into the membrane of the endoplasmic reticulum (ER) and targets newly-synthesized CD4 for degradation in cytosolic proteasomes. The mechanism of this targeting was thought to be distinct from the canonical ER-associated degradation (ERAD) pathway. However, in collaboration with Klaus Strebel (NIAID) and Yihong Ye (NIDDK), we recently discovered that CD4 downregulation by Vpu involves at least part of the ERAD pathway. In particular, the p97-Npl4-Ufd1 ERAD dislocase is required for Vpu-induced CD4 targeting for proteasomal degradation. We also found that targeting requires ubiquitination of CD4 on not only lysine but also serine and threonine residues. Finally, we demonstrated that Vpu induces CD4 retention in the ER in addition to ERAD targeting. This retention is in large part dependent on transmembrane domain interactions. Together, Nef and Vpu ensure profound and sustained suppression of CD4 expression throughout the HIV-1 infectious cycle.